The goal of the proposed research is to design a new class of in vitro and in vivo biosensors in which rapid, analyte-triggered droplet fusion creates a uniquely detectable acoustic signal. Development of a technology that could provide a convenient, inexpensive, and portable method for detecting biomolecules without sample manipulation would provide new avenues for measuring both systemic and localized biomolecular levels in many different environments and media. For in vitro detection, an in-solution sensor would obviate the need for sample processing and washing steps that may lead to added time, expense, and sources of error. In addition, droplets can be utilized to almost any scale, from microfluidic chips to batch processing. Finally, the almost nonexistent acoustic background in controlled in vitro environments allows this method to bypass problems with background noise, such as autofluorescence, that greatly complicate and mitigate signals arising from typical biomarker detection modalities. For in vivo imaging, there are few technologies that can respond to levels of specific biomarkers in a localized environment, and those that can typically possess only modest on-off ratios. Building from our previous success with biochemically-responsive ultrasound contrast agents, the proposed sensors will create specifically detectable ultrasound signals for in vivo imaging that we expect will exceed our previous benchmark of 20 dB (100-fold) on-off ratios. The proposed research will mark the first example of sound as a chemical detection modality, which would facilitate the construction of an entirely new class of sensors, diagnostics, and imaging agents. We propose to perform single and multiplexed sound sensing by employing biomarker-driven generation of microbubbles, which scatter sound in a unique way that cannot be found in any biological medium. Because of the small expense associated with both the proposed detection reagents and system, the proposed technology is expected to be useful for both routine clinical analyses and point-of-care diagnostics. To generate microbubbles only in response to biomolecular analytes, we propose to create emulsions that are poorly visible to ultrasound under normal conditions but transform into bubbles upon sensing a specific biomolecule. The emulsions will be formulated with oligonucleotides and peptide epitopes that will promote fusion through biochemical recognition. Once the emulsions reach a critical size they may be specifically vaporized into bubbles by sound or controlled heating, even in the presence of unactivated emulsions. By employing a mechanism by which sensing analytes either block or permit the formation of detectable droplets, this technology can be applied to both in vitro diagnostics and in vivo imaging.